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. 2017 Oct 10;8(55):94462–94480. doi: 10.18632/oncotarget.21776

Figure 2. Confirmation of mutant viruses.

Figure 2

(A) MDBK cells were infected with each virus at a MOI of 3. PCR were run with the indicated viral DNAs of pUL21 using the UL21 F/R primers (Table 1) and pUL35 using the UL35 F/R primers (Table 1). (B) Expression of pUL21 recombinant viruses was confirmed by RT-PCR with the indicated viral cDNA using primers UL21 F/R (Table 1) via agarose gel electrophoresis. The bovine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as the internal reference control. DNA ladders are shown in bp to the left of the gel. (C) MDBK cells were infected with indicated viruses at a MOI of 3. Cells were harvested at 18 hpi and separated on a 15% SDS-PAGE and transferred to a PVDF membrane. Then, the membranes were probed sequentially with a mouse anti-HA antibody, followed by HRP-conjugated goat anti-mouse IgG. Beta-actin was used as the internal reference control. (D) MDBK cells were infected at a MOI of 3 with the indicated viruses. At 18 hpi, cell lysates were electrophoresed through 12% polyacrylamide gels and transferred onto PVDF membranes. Membranes were probed with the UL21 antisera. The molecular weights of the protein markers are shown in kDa to the left of the gel. (E) RFLP analysis obtained by restriction enzyme HindIII genomic digestion of UL21 mutant, vBoHV-1-∆UL21R and BoHV-1 viruses.